Methods and apparatus for magnetic multi-bead assays

ABSTRACT

The present application discloses methods and apparatus for detecting a complex including an analyte that include contacting a sample in a solution with a population of functionalized beads of a first type, which are magnetic functionalized beads and are functionalized to include a first moiety that associates with an analyte under suitable conditions, contacting the sample solution with a population of functionalized beads of a second type, which are functionalized to include a second moiety that associates with the analyte under suitable conditions, contact resulting in formation of a complex including one of the first type of functionalized bead, the analyte, and one of the second type of functionalized bead, and detecting the complex including the analyte by detecting magnetic fields produced by the magnetic functionalized bead and by detecting the functionalized bead of the second type associated with the analyte in the complex.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application based on U.S. patentapplication Ser. No. 16/467,474 filed on Jun. 6, 2019 entitled METHODSAND APPARATUS FOR MAGNETIC MULTI-BEAD ASSAYS, which in turn is anational stage application, filed under 35 U.S.C. § 371, ofInternational Patent Application No. PCT/US17/68126 filed on Dec. 22,2017 entitled METHODS AND APPARATUS FOR MAGNETIC MULTI-BEAD ASSAYS,which claims the benefit of U.S. Provisional Patent Application No.62/438,593 filed on Dec. 23, 2016 entitled METHODS AND APPARATUS FORMAGNETIC MULTI-BEAD ASSAYS. The entire contents of the aboveapplications are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under contract numberHR0011-14-C-0020 awarded by the Defense Advanced Research ProjectsAgency of the Department of Defense. The government has certain rightsin the invention.

BACKGROUND

Enzyme linked immunosorbent assay (ELISA) has been an industry widestandard research technique used for measuring protein analytes frombiological matrices since its introduction in the 1960s. In its basicconception, two antibodies (immunoglobulins) are used to capture asingle protein analyte. The resulting immunocomplex is identified andmeasured using an enzyme and reporter buffer. The enzyme is typicallybound to one of the antibodies through covalent binding. The enzyme,when incubated in the presence of the reporter buffer, converts thesubstrate to a functional reporter which can be measured analytically byspectrophotometric means.

Current state of the art ELISA-related technologies are replacing theplate-based format with a single-bead-based format. Single-bead-basedELISAs have one antibody that is bound to a solid surface, typically abead, and the second antibody is labeled with biotin. The capture beadcontains material that allows it to be easily manipulated by an appliedmagnetic field, including separating the bead and any analytes bound tothe bead from a sample suspension. This process, called magneticseparation, is well known in the art and may be used to concentrate thetarget analyte and to remove unbound material such as unwanted proteinsthat may contribute to signal background. Compared to traditionalELISAs, single-bead-based ELISAs can provide improved sensitivity totarget analytes with lower background and in a shorter amount of time.

Nevertheless, there is a need for continuing improvement in sensitivityand specificity to target analytes.

BRIEF SUMMARY

Various embodiments disclosed herein relate to methods and apparatus fordetecting a complex including an analyte in a sample by observingcomplexes containing two or more distinguishable beads (i.e., beads ofdifferent types) that are bound by the analyte. In accordance with oneor more embodiments, a bead-based magnetic assay system for detecting acomplex including an analyte based on optically detected magneticresonance (ODMR) includes a plurality of functionalized beads of a firsttype, which are magnetic functionalized beads and are functionalized toinclude a first moiety that associates with an analyte under suitableconditions, a plurality of functionalized beads of a second type, whichare functionalized to include a second moiety that associates with theanalyte under suitable conditions, a substrate including at least oneODMR center, a light source configured to generate incident light thatexcites electrons within the at least one ODMR center from a groundstate to an excited state, a magnet for applying a bias magnetic fieldon a complex disposed over the at least one ODMR center, the complexincluding one of the first type of functionalized bead, the analyte, andone of the second type of functionalized bead. The system furtherincludes a microwave source configured to generate a microwave fieldincident on the at least one ODMR center, the microwave source beingfurther configured to generate the microwave field with frequencies thatcorrespond to ground state transitions in the at least one ODMR center,in which the at least one ODMR center produces emitted light whenilluminated by the incident light, characteristics of the emitted lightbeing influenced by the microwave field and by the magneticfunctionalized bead associated with the analyte in the complex, and anoptical photodetector that detects light emitted by the at least oneODMR center. In some embodiments, the at least one ODMR center can be asilicon vacancy center in a silicon carbide lattice. In otherembodiments, the at least one ODMR center can be a silicon vacancycenter in a diamond lattice. In still other embodiments, the at leastone ODMR center can be a nitrogen-vacancy center in a diamond lattice.In certain embodiments, the at least one ODMR center can be formed in anupper surface of the substrate. In some embodiments, the at least oneODMR center can be a plurality of ODMR centers formed in the uppersurface of the substrate. In these embodiments, the opticalphotodetector can be an optical imaging system having an imaging sensorthat images the emitted light from the plurality of ODMR centers. Incertain embodiments, each of the first and the second moiety can be areceptor, protein, antibody, cell, virus, or nucleic acid sequence. Insome embodiments, the functionalized beads of the first type can besuperparamagnetic functionalized beads including a superparamagneticmaterial. In certain embodiments, the functionalized beads of the firsttype can include a nonmagnetic layer encapsulating the superparamagneticmaterial. In some embodiments, the superparamagnetic functionalizedbeads can include iron oxide particles. In certain embodiments, thefunctionalized beads of the first type can comprise magneticnanoparticles disposed within a polymer substrate. In other embodiments,the functionalized beads of the first type can comprise magneticnanoparticles disposed on a surface of a polymer substrate. In someembodiments, the functionalized beads of the second type can befluorescent functionalized beads. In other embodiments, thefunctionalized beads of the second type can be magnetic functionalizedbeads including a quantity of magnetic material distinguishable from thefunctionalized beads of the first type. In still other embodiments, thefunctionalized beads of the second type can be magnetic functionalizedbeads, the second type of functionalized beads including a magneticproperty distinguishable from the functionalized beads of the firsttype. In some embodiments, the functionalized beads of the first typecan be superparamagnetic functionalized beads including asuperparamagnetic material. In certain embodiments, the functionalizedbeads of the first type can include a nonmagnetic layer encapsulatingthe superparamagnetic material. In some embodiments, the functionalizedbeads of the second type can be ferromagnetic functionalized beadsincluding a ferromagnetic material. In certain embodiments, thefunctionalized beads of the second type can include a nonmagnetic layerencapsulating the ferromagnetic material. In some embodiments, each ofthe first type of functionalized beads and the second type offunctionalized beads can have a diameter in a range of between 50 nm and10 μm. In certain embodiments, each of the diameters of thefunctionalized beads of the first type and the second type can be in arange of between 0.5 μm and 5 μm. In some embodiments, the diameter ofthe functionalized beads of the first type can be similar to thediameter of the functionalized beads of the second type. In otherembodiments, the diameter of the functionalized beads of the first typecan be different from the diameter of the functionalized beads of thesecond type by at least 50%. In some embodiments, the system can furtherinclude a plurality of functionalized beads of at least a third type,functionalized to include at least the second moiety that associateswith at least a second analyte under suitable conditions. In certainembodiments, the system can further include a plurality offunctionalized beads of a fourth type, functionalized to include thesecond moiety that associates with the second analyte under suitableconditions. In some embodiments, the functionalized beads of the firstand/or second type can further include at least one additional moietythat associates with the second analyte under suitable conditions. Incertain embodiments, the system can further include a third moiety thatassociates with a third analyte under suitable conditions, wherein thefunctionalized beads of the first type are further functionalized toinclude the second moiety, and the functionalized beads of the secondtype are further functionalized to include the third moiety.

In accordance with one or more embodiments, a method of detecting acomplex including an analyte includes contacting a sample in a solutionwith a population of functionalized beads of a first type, which aremagnetic functionalized beads and are functionalized to include a firstmoiety that associates with an analyte under suitable conditions,contacting the sample solution with a population of functionalized beadsof a second type, which are functionalized to include a second moietythat associates with the analyte under suitable conditions, contactresulting in formation of a complex including one of the first type offunctionalized bead, the analyte, and one of the second type offunctionalized bead, and detecting the complex including the analyte bydetecting magnetic fields produced by the magnetic functionalized beadand by detecting the functionalized bead of the second type associatedwith the analyte in the complex. In some embodiments, the method canfurther include disposing the sample solution including the complex overa substrate that includes at least one optically detected magneticresonance (ODMR) center formed in the substrate, exciting electronswithin the at least one ODMR center from a ground state to an excitedstate with incident light, applying a bias magnetic field on thecomplex, and generating a microwave field incident on the at least oneODMR center, the microwave field including frequencies that correspondto ground state transitions in the at least one ODMR center, whereindetecting the complex including the analyte further includes analyzinglight emitted by the at least one ODMR center, characteristics of theemitted light being influenced by the microwave field and by themagnetic functionalized bead associated with the analyte in the complex.In some embodiments, the at least one ODMR center can be anitrogen-vacancy center in a diamond lattice. In certain embodiments,the at least one ODMR center can be formed in an upper surface of thesubstrate. In some embodiments, the at least one ODMR center can be aplurality of ODMR centers formed in the upper surface of the substrate.In these embodiments, analyzing light emitted by the plurality of ODMRcenters includes imaging the emitted light. In certain embodiments, themethod can further include applying a magnetic field gradient to thesample solution after contacting the sample with the population offunctionalized beads of the first type. In some embodiments, applyingthe magnetic field gradient to the sample solution can be performedafter contacting the sample solution with the population offunctionalized beads of the second type. In certain embodiments, thepopulation of functionalized beads of the first type and the populationof functionalized beads of the second type can be added to the samplesolution sequentially. In some embodiments, the functionalized beads ofthe second type can be fluorescent functionalized beads, and the methodcan further include illuminating the complex with incident light thatexcites fluorescence within the functionalized beads of the second typeand fluorescence imaging of the complex. In other embodiments, thefunctionalized beads of the second type can be magnetic functionalizedbeads, including a magnetic property distinguishable from thefunctionalized beads of the first type. In some embodiments, the methodcan further include applying a magnetic field gradient to the samplesolution after contacting the sample solution with the functionalizedbeads of the first and second types. In certain embodiments, the methodcan further include varying the magnetic field gradient applied to thesample solution. In some embodiments, the method can further includeconcentrating the sample solution after contacting the sample solutionwith the population of functionalized beads of the second type. Incertain embodiments, the method can further include agglomerating aplurality of functionalized beads of the first and second types, aftercontacting the sample solution with the population of functionalizedbeads of the second type, before detecting the complex. In someembodiments, the method can further include dehydrating the samplesolution after disposing the sample solution over the diamond substrate.

In accordance with one or more embodiments, a bead-based assay systemfor detecting a complex including an analyte includes a plurality offunctionalized beads of a first type, which are magnetic functionalizedbeads and are functionalized to include a first moiety that associateswith an analyte under suitable conditions, a plurality of functionalizedbeads of a second type, which are fluorescent functionalized beads, andare functionalized to include an unlabeled moiety that associates withthe analyte under suitable conditions, a light source configured togenerate incident light that excites fluorescence within thefunctionalized beads of the second type, and an optical fluorescencedetector that detects fluorescence emitted by the functionalized beadsof the second type associated with the analyte in a complex includingone of the first type of functionalized bead, the analyte, and one ofthe second type of functionalized bead. In certain embodiments, thefluorescent functionalized beads can comprise a polymer substrateimpregnated with a fluorescent material. In some embodiments, theoptical fluorescence detector can include a spectrophotometer. In otherembodiments, the optical fluorescence detector can include an opticalimaging sensor that images the fluorescence emitted by thefunctionalized beads of the second type associated with the analyte inthe complex. In some embodiments, the functionalized beads of the firsttype can be superparamagnetic functionalized beads. In certainembodiments, the superparamagnetic functionalized beads can include ironoxide particles. In some embodiments, the functionalized beads of thefirst type can include magnetic nanoparticles disposed within thepolymer substrate. In other embodiments, the functionalized beads of thefirst type can include magnetic nanoparticles disposed on a surface ofthe polymer substrate.

In accordance with one or more embodiments, a method of detecting acomplex including an analyte includes contacting a sample in a solutionwith a population of functionalized beads of a first type, which aremagnetic functionalized beads and are functionalized to include a firstmoiety that associates with an analyte under suitable conditions,contacting the sample solution with a population of functionalized beadsof a second type, which comprise a polymer substrate impregnated with afluorescent material, and are functionalized to include an unlabeledmoiety that associates with the analyte under suitable conditions,contact resulting in formation of a complex including one of the firsttype of functionalized bead, the analyte, and one of the second type offunctionalized bead, illuminating the complex with incident light thatexcites fluorescence within the functionalized beads of the second type,and detecting the complex including the analyte by analyzingfluorescence emitted by the functionalized beads of the second typeassociated with the analyte in the complex. In some embodiments, themethod can further include applying a magnetic field gradient to thesample solution after contacting the sample with the population offunctionalized beads of the first type. In certain embodiments, applyingthe magnetic field gradient to the sample solution can be performedafter contacting the sample solution with the population offunctionalized beads of the second type. In some embodiments, the methodcan further include concentrating the sample solution after contactingthe sample solution with the population of functionalized beads of thesecond type, before detecting the complex. In certain embodiments, themethod can further include agglomerating a plurality of functionalizedbeads of the first and second types, after contacting the samplesolution with the population of functionalized beads of the second type,before detecting the complex. In some embodiments, the method canfurther include dehydrating the sample solution before detecting thecomplex.

Magnetic multi-bead assays improve upon bead-based ELISAs by detectingtarget analytes bound in complexes, such as immunocomplexes. Bycombining the convenience and simplicity of magnetic separation withrobust and sensitive detection of beads, magnetic multi-bead assaysprovide excellent sensitivity with a simple, rapid process.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments, as illustrated in the accompanyingdrawings in which like reference characters refer to the same partsthroughout the different views. The drawings are not necessarily toscale, emphasis instead being placed upon illustrating embodiments.

FIG. 1A schematically illustrates a functionalized bead of a first type,a first moiety, a functionalized bead of a second type, a second moiety,and an analyte in accordance with one or more embodiments.

FIG. 1B schematically illustrates a complex that includes one of afunctionalized bead of a first type, a first moiety, one of afunctionalized bead of a second type, a second moiety, and an analyte inaccordance with one or more embodiments.

FIG. 1C schematically illustrates a bead-based assay system fordetecting a complex including a spectrophotometer that detectsfluorescence emitted by the functionalized beads of the second type inaccordance with one or more embodiments.

FIG. 1D illustrates a plot of relative fluorescence units (RFU) as afunction of PSA (pM) in accordance with one or more embodiments.

FIG. 2 illustrates a method of detecting a complex including an analytein accordance with one or more embodiments.

FIG. 3A schematically illustrates a bead-based assay system fordetecting a complex including an optical imaging sensor that imagesfluorescence emitted by the functionalized beads of the second type inaccordance with one or more embodiments.

FIG. 3B illustrates a fluorescent image in accordance with one or moreembodiments.

FIG. 3C illustrates a plot of signal as a function of PSA concentration(pM) in accordance with one or more embodiments.

FIG. 4 illustrates another method of detecting a complex including ananalyte in accordance with one or more embodiments.

FIG. 5 schematically illustrates a wide-field diamond magnetic imagingapparatus in accordance with one or more embodiments.

FIG. 6A schematically illustrates several complexes including magneticbeads and fluorescent beads in accordance with one or more embodiments.

FIG. 6B illustrates a fluorescent image of the complexes shown in FIG.6A in accordance with one or more embodiments.

FIG. 6C illustrates a magnetic image of the complexes shown in FIG. 6Ain accordance with one or more embodiments.

FIG. 7A schematically illustrates a complex that includes a magneticfunctionalized bead of a first type, a magnetic functionalized bead of asecond type, and an analyte in accordance with one or more embodiments.

FIG. 7B schematically illustrates a complex including magnetic beads ofthe first and second types in accordance with one or more embodiments.

FIG. 7C illustrates a positive magnetic image of the magnetic beadsshown in FIG. 7B in accordance with one or more embodiments.

FIG. 7D illustrates a negative magnetic image of the magnetic beadsshown in FIG. 7B in accordance with one or more embodiments.

FIG. 8A illustrates magnetic bead discrimination based on remanence andsusceptibility in accordance with one or more embodiments.

FIG. 8B illustrates magnetic bead discrimination based on magnetizationmagnitude in accordance with one or more embodiments.

FIG. 8C illustrates magnetic bead discrimination based on magneticanisotropy in accordance with one or more embodiments.

FIG. 8D illustrates magnetic bead discrimination based on magneticcoercivity in accordance with one or more embodiments.

FIG. 8E illustrates magnetic bead discrimination based on AC magneticresponse in accordance with one or more embodiments.

FIG. 8F illustrates magnetic bead discrimination based on magneticsaturation in accordance with one or more embodiments.

FIG. 9 illustrates a magnetic image of two beads of different sizes inaccordance with one or more embodiments.

FIGS. 10A-1, 10B-1, and 10C-1 illustrate magnetic images of the imagesignal of three beads B in accordance with one or more embodiments.

FIGS. 10A-2, 10B-2, and 10C-2 illustrate magnetic images of thedifference signal after subtracting the characteristic signal of threebeads B in accordance with one or more embodiments.

FIGS. 11A-1, 11B-1, and 11C-1 illustrate magnetic images of the imagesignal of three dimers of beads A and B in accordance with one or moreembodiments.

FIGS. 11A-2, 11B-2, and 11C-2 illustrate magnetic images of thedifference signal of three dimers beads A and B after subtracting thecharacteristic bead B signal in accordance with one or more embodiments.

FIG. 12A schematically illustrates a multiplexed assay including fourdistinguishable bead types in accordance with one or more embodiments.

FIG. 12B schematically illustrates complexes including fourdistinguishable bead types in accordance with one or more embodiments.

FIG. 13A schematically illustrates a multiplexed assay including threedistinguishable bead types in accordance with one or more embodiments.

FIG. 13B schematically illustrates complexes including threedistinguishable bead types in accordance with one or more embodiments.

DETAILED DESCRIPTION

As stated above, various embodiments disclosed herein relate to methodsand apparatus for detecting a complex including an analyte in a sampleby observing complexes containing two or more distinguishable beads(i.e., beads of different types) that are bound by the analyte. At leastone of the bead types is magnetic and can be concentrated in a liquidsuspension by means of magnetic forces exerted with an applied magneticfield. A fully-magnetic assay may also be implemented by using diamondmagnetic imaging to distinguish between magnetic bead types withdistinct magnetic properties.

In accordance with one or more embodiments, wide-field magnetic imagingusing nitrogen-vacancy (NV) centers in diamond can be used to provide aplatform on which to implement a multi-bead assay that is fullymagnetic, eliminating the need to detect fluorescence from the sample.Magnetic beads provide strong, stable signals by means of the magneticfields they produce, which permeate through biological sample matricesand contaminants and allow for unambiguous detection. Magnetic beads canfurther be manipulated to accelerate assay kinetics and enable rapidsample preparation with minimal hardware. The fully magnetic assay canbe deployed with a small-footprint instrument and low reagent volumes,delivering rapid assay results at low cost.

Magnetic Multi-Bead Assays

Magnetic multi-bead assays make use of distinct bead types to determineanalyte concentration in a sample by detecting the formation of beadcomplexes that are bound by the analyte. One bead type has magneticproperties that allow for separation of beads and bound material from asuspension of beads in liquid with the application of a magnetic fieldgradient. This process, called magnetic separation, is commonly used toisolate or concentrate target analytes, including cells, proteins, andnucleic acids.

The sensitivity of the magnetic multi-bead assay stems in part fromthree features:

-   -   (1) The assay measures co-presence of at least two        distinguishable beads, such that detection of the target analyte        only results from the analyte binding to at least two distinct        antibodies on at least two distinguishable bead types. This        assay provides enhanced target specificity through the combined        specificity of multiple antibodies, which in turn provides        better sensitivity.    -   (2) Confounding effects, such as signal backgrounds, caused by        sample components other than the target analyte can be reduced        or eliminated by purifying the sample using magnetic separation.    -   (3) Beads can be detected rapidly and with high accuracy and        precision. Bead signals can be stronger and more stable and can        be detected more quickly than signals from molecular reporters        including fluorescent dyes and fluorescent products of enzymatic        activity.

Complex Formation

To measure a target analyte in a multi-bead assay, the analyte must bindto at least two distinguishable beads to form a complex, so that thepresence of both beads can be detected. The beads may be coated withbinding ligands, herein also denoted as moieties, such as antibodies,that bind specifically to and thereby associate with a certain region ofa certain target analyte. Each bead in the multi-bead assay may becoated with one or more different types of binding ligands. Differentbead types used in the multi-bead assay may have the same binding ligandtypes, overlapping sets of binding ligand types, or distinct bindingligand types. In the simplest case, two distinct bead types areused—hereafter denoted bead A and bead B. (In other embodiments, threeor more distinct bead types can be used.) In the two-bead example, beadA and bead B are coated with antibodies, with antibody X on bead A andantibody Y on bead B. Antibody X binds specifically to a differentregion of the target analyte than antibody Y so that the target analytemay be bound to both simultaneously.

The complex, such as an immunocomplex, may be formed under suitableconditions, such as by incubating the sample with a suspension of bead Aand bead B. Target analytes in the sample will encounter a bead surfaceas they diffuse through the sample, and bind to it. The sample may bemixed, shaken, or otherwise agitated to accelerate this process. As thebeads also move through the sample, they will encounter analytes boundto beads of the opposite type and will additionally bind to thoseanalytes, forming heterogeneous bead complexes of the form A-B, A-B-A,B-A-B, and other combinations.

The beads and bead complexes are then concentrated together by magneticseparation. First, a magnetic field gradient is applied that exerts amagnetic force on bead A. Any bead A and bead complex containing bead Awill be separated from the sample. In some embodiments, bead B may alsobe magnetic and experience a similar magnetic force, forming a “pellet”of magnetic material and bound analytes. Unbound sample components,referred to here as “background material,” will not be separated andtherefore may be discarded with the supernatant above the pellet. Themagnetic gradient may then be removed and the beads may be re-suspendedin the same or different buffer solution. This process may be repeatedto reduce the concentration of background material. Magnetic separationmay be performed by hand or automated with a commercial plate washer.

Alternately, complex formation can also be performed in discrete steps,which may reduce signal background caused by nonspecific binding ofbeads into complexes in the absence of the target analyte. Bead A mayfirst be added to the sample to capture the target analyte, followed bymagnetic separation to reduce the concentration of background material.Bead B can then be added separately to this purified sample. Whetherbead A and bead B are added together or sequentially will be determinedempirically and will depend on the antibodies utilized and whethernonspecific binding is significantly reduced using sequential bindingsteps.

If bead B is less magnetic than bead A, or nonmagnetic, then magneticseparation may be used after forming complexes to reduce signalbackground associated with unbound bead B. For example, a less-magneticbead B will be separated from the sample suspension more slowly thanbead A, so that magnetic separation can be terminated at a point atwhich bead A has been suitably separated into a pellet while theseparation of bead B remains incomplete. If at this point thesupernatant above the pellet is discarded, a significant fraction ofbead B will be removed, but bead A will be preserved, includingcomplexes containing bead A.

In accordance with one or more embodiments, the diameters of bead A andbead B may be in a range of between 50 nm and 10 μm, such as between 0.5μm and 5 μm. Bead A and bead B may be chosen to have differentdiameters, such that the diameter of the functionalized beads of thefirst type is different from the diameter of the functionalized beads ofthe second type by at least 50%, so that the two bead types may bedistinguished by the spatial distribution of their respective magneticfield signals. Alternatively bead A and bead B may be chosen to havesimilar diameters, that is, diameters different by less than 50%, sothat they exhibit similar surface area, move similarly in the liquidsample suspension, occupy a similar amount of space in the detectionregion, and provide similar signal magnitudes. Bead diameters in therange of 0.5 μm to 5 μm may allow for rapid magnetic separation (in amatter of seconds) and a large quantity of binding ligands on each bead.In addition, bead diameters in this range are similar to or slightlylarger than the typical diffraction-limited imaging resolution of anoptical microscope or wide-field diamond magnetic imaging system.

Complex Detection

Once complexes, such as immunocomplexes, have been formed (heterogeneousbead complexes containing bead A and bead B bound by the analyte), theyare measured by detecting the co-presence of both bead types. Severalmethods of detecting the co-presence of both bead types that can beimplemented to achieve this goal are described further below.

The measurement of complexes containing the analyte can be calibratedwith a range of calibration samples of known analyte concentration sothat a given measurement of complexes implies a certain analyteconcentration. The measurements of the range of calibration samples iscollectively referred to as a calibration curve. Detection of complexes,by the methods and apparatus described herein, enables measuring analyteconcentration in combination with a calibration curve.

Example A1: Magnetic-Fluorescent Assay with Plate Reader

Consider bead A to be superparamagnetic, composed of magneticnanoparticles of a superparamagnetic material dispersed within or on thesurface of a polymer substrate. Suitable superparamagnetic materialsinclude, for example, iron oxide, Fe₂O₃ or Fe₃O₄, manganese ferrites(MnFe₂O₄), or cobalt ferrites (CoFe₂O₄), in the form of single crystalnanoparticles less than about 20 nm in size, typically in a range ofbetween 5 nm and 10 nm. The magnetic nanoparticles are engineered to besmall enough that they exhibit no remanent magnetization in the absenceof an applied field (superparamagnetism). When a field is applied, theparticles magnetize in the direction of the field, producing a beadmagnetization sufficient for magnetic separation.

Consider bead B to be a fluorescent functionalized bead including apolymer substrate impregnated with fluorescent material. Bead B isnonmagnetic, such that unbound bead B is left behind during magneticseparation.

As shown in FIGS. 1A-1C, a bead-based assay system 100 for detecting acomplex including an analyte, shown in FIG. 1C, includes, as shown inFIG. 1A, a plurality of functionalized beads 110 of a first type (beadA), which are magnetic functionalized beads and are functionalized toinclude a first moiety X 115 that associates with an analyte 120 undersuitable conditions, a plurality of functionalized beads 130 of a secondtype (bead B), which are fluorescent functionalized beads, and arefunctionalized to include an unlabeled moiety Y 135 that associates withthe analyte 120 under suitable conditions, and, as shown in FIG. 1C, anoptical photodetector 140 that detects light 155 emitted by thefunctionalized beads 130 of the second type associated with the analyte120 in a complex 145, shown in FIG. 1B, including the first type offunctionalized bead 110, the analyte 120, and the second type offunctionalized bead 130. As shown in FIG. 1C, the optical photodetector140 is a spectrophotometer 140 that detects fluorescence 155 emitted bythe functionalized beads 130 of the second type that fluoresce whenilluminated by light from a filtered lamp 150. Each of the first 115 andsecond 135 moiety can be a receptor, protein, antibody, cell (eukaryoticor prokaryotic), organelle, virus, or nucleic acid sequence. The secondmoiety 135 is unlabeled, that is, not labeled with a fluorophore. Insome embodiments, the fluorescent functionalized beads can comprise apolymer substrate impregnated with a fluorescent material. Severalimprovements arise from having the fluorescent material impregnatedinside the functionalized beads B and using an unlabeled second moiety135. First, the volume of the bead allows for much greater quantities offluorophores to be included and measured, as compared to surfaceattachment, because the volume of the bead is significantly greater thanits surface area. Second, in other methods whereby the surface boundmoiety is labeled with a fluorophore, the quantity of fluorophore isfurther reduced as the moieties do not cover the entire surface,resulting in even further reduced labeling. Third, fluorophores aresensitive to light, temperature, pH, salt and other environmentalconditions associated with biological assays. As such, the fluorophoresimpregnated into the bead are sheltered and protected from the chemicalenvironment, which results in brighter and more robust detection.Fourth, for fluorophores to be conjugated or covalently bound to surfaceattached moieties, they must undergo a chemical reaction that can alterthe state of the fluorophore (such as its 3D structure, charge,polarity) that can negatively affect the function of the fluorophore.

Due to the superparamagnetic nature of the capture bead A, extra bead Awill be included in the final read. However, since this bead is in adifferent fluorescent channel (wavelength) or not fluorescent at all, itwill not negatively affect the positive signal of the detector bead B orprovide additional non-specific (fluorescent) background.

In one embodiment, the magnetic multi-bead assay is performed asdescribed below. After complex formation, a magnetic separation step orseries of repeated steps is used to reduce the unbound bead Bpopulation. After magnetic separation, the continued presence of bead Bindicates successful binding of the target analyte both to bead A and tobead B—otherwise either the analyte or bead B or both would likely havebeen discarded during magnetic separation. Detecting bead B in thesample suspension is therefore sufficient to establish co-presence ofboth beads in complexes.

Accordingly, as shown in FIG. 2 , a method 200 of detecting a complexincluding an analyte includes contacting 210 a sample in a solution witha population of magnetic functionalized beads of a first type,contacting 220 the sample solution with a population of fluorescentfunctionalized beads of a second type, illuminating 230 the complex withincident light that excites fluorescence within the functionalized beadsof the second type, and detecting 240 the complex including the analyteby analyzing the fluorescence.

The sample suspension may be analyzed using a fluorescent plate readeror similar device that uses a spectrophotometer to optically excite andmeasure fluorescence from each plate well. The suspension may betransferred to low-fluorescence black plates prior to measurement toreduce signal background produced by the plate. While it is possiblethat the reactions could be performed in the black plates originally,reactions may be more efficiently performed in round-bottom plates thatmay be unavailable in black plastic. During the fluorescencemeasurement, bead B fluorescence may be both induced and recordedthrough optical band pass filters. A titration of bead B and a wellcontaining no bead B may be separately measured to calibrate theobserved fluorescence signal to a known bead concentration under similarbuffer conditions.

Most commercially available fluorescent plate readers can be configuredwith standard excitation and emission filters, dichroic or band passfilters, and proper gain settings or photo multiplier tube (PMT)adjustments to satisfactorily depress autofluorescence of sample bufferand amplify true fluorescent signal from the ensemble fluorescent beads.The wavelength of measured fluorescence will depend upon the choice offluorophore incorporated into the bead. It should be noted that lowfluorophore concentration or weak fluorophores may depress thefluorescent signal and reduce the sensitivity of the assay.

As shown in FIG. 1D, prostate specific antigen (PSA) as low as 0.1 pg/mLcan be measured with little optimization of the plate reader conditions.As few as 250 fluorescent beads can be measured at the minimum signallevel, considered to be the fluorescence measurement background meanplus triple its standard deviation. Longer read times, changes inphotomultiplier tube (PMT) gain settings, or adjustments in the scannedregion of each well all may contribute to improved sensitivity.Optimization of these parameters depends upon the features of any givenplate reader.

Example B2: Magnetic-Fluorescent Assay with Fluorescence Imaging

Consider bead A and bead B to be of the same types described in ExampleA1 above. Further consider that, as in Example A1, a final magneticseparation step or series of steps is performed to reduce theconcentration of bead B.

In another embodiment employing a bead-based assay system 300, shown inFIG. 3A, bead B fluorescence may be measured by fluorescence imagingrather than with a spectrophotometer. The sample suspension or a portionof it may be dispersed on a microscope slide 305. Under appropriateoptical excitation, bead B fluorescence may be imaged by a microscopysystem onto a camera sensor 340 through an optical band pass filter 348that blocks the excitation light from the filtered lamp 150. In theresulting bead B fluorescence image, and provided suitable resolution ofthe microscopy system, individual beads may be resolved, identified, andcounted (as illustrated in FIG. 3B). The total number of beads B countedin the image provides a measurement of the number of analytes present inthe sample, since the observation of bead B fluorescence 155 impliesco-presence of both bead A and bead B bound to the target analyte in thecomplex 145.

Fluorescence imaging may provide an improvement in sensitivity above theplate reader measurement described above in Example A1. This improvementarises from the ability to reject confounding signals, including:

-   -   (1) optical detector backgrounds, such as arise from optical        filter leakage and optical sensor noise;    -   (2) diffuse fluorescence backgrounds, such as autofluorescence        from buffer components;    -   (3) fluorescence from contaminants, such as dust particles, that        may be clearly distinguished in images from bead B signals.

Rejecting false signals allows for a lower signal background, as shownin FIG. 3C, where the imager 340 yields a lower signal level for thesame PSA concentration as compared to the plate reader 140, and acorrespondingly improved sensitivity to low complex concentrations thatresult from low analyte concentrations.

Imaging of bead B may be performed with a liquid sample suspension, suchas a droplet on a microscope slide under a coverslip, or after drying arepresentative droplet of the liquid sample. After drying, fluorescentbead B remains bright and no longer moves under diffusion or due to flowof the sample on the slide, which enables longer exposure times andlower excitation light intensity. The buffer solution is chosen topreserve immunocomplexes against dissociation during drying, to dispersebeads relatively uniformly over the dried region, and to avoid leavingsolute crystals or other residue that may impede imaging.

Example C3: Magnetic-Fluorescent Assay with Magnetic and FluorescenceImaging

Consider bead A and bead B to be of the same types described in ExampleA1 above. Further consider that, as in Example A1, a final magneticseparation step or series of steps is performed to reduce theconcentration of bead B and that bead B is counted by imaging the samplewith a fluorescence microscopy system.

In accordance with one or more embodiments, as shown in FIG. 4 , amethod 400 of detecting a complex including an analyte includescontacting 410 a sample in a solution with a population of magneticfunctionalized beads of a first type, contacting 420 the sample solutionwith a population of functionalized beads of a second type, anddetecting 430 the complex including the analyte by detecting magneticfields produced by the magnetic functionalized bead and by detecting thefunctionalized bead of the second type associated with the analyte inthe complex. In some embodiments, detecting magnetic fields includesusing any magnetic imaging technology, such as magnetic force microscopyor a scanning Hall probe. In certain embodiments, detecting thefunctionalized beads of the second type includes detecting fluorescenceas described in Examples A1 or B2 above.

In another embodiment, the microscopy system may include a wide-fielddiamond magnetic imaging system that allows for imaging of bead A, whichis superparamagnetic. Wide-field diamond magnetic imaging withnitrogen-vacancy (NV) centers in diamond is capable of rapidly imagingmagnetic fields disposed over the surface of a diamond sensor, at roomtemperature, with sub-micron resolution. Magnetic images may beco-registered to conventional optical fluorescence or bright-fieldimages acquired for the same field of view with the same imaging system.Adjustments to the imaging system may be made between magnetic andoptical imaging to optimize performance, such as changing opticalfilters or correcting focal position.

As shown in FIG. 5 , a bead-based magnetic assay system 500 fordetecting a complex including an analyte based on optically detectedmagnetic resonance (ODMR) includes, as shown in FIG. 1A and describedabove, a plurality of functionalized beads 110 of a first type, whichare magnetic functionalized beads and are functionalized to include afirst moiety 115 that associates with an analyte 120 under suitableconditions, a plurality of functionalized beads 130 of a second type,which are functionalized to include a second moiety 135 that associateswith the analyte 120 under suitable conditions, and, as shown in FIG. 5, a substrate 532 including at least one ODMR center 540 (a plurality ofODMR centers 540 shown in FIG. 5 ), a light source 536 configured togenerate incident light that excites electrons within the at least oneODMR center 540 from a ground state to an excited state, a magnet 534for applying a bias magnetic field on a complex 530 disposed over the atleast one ODMR center 540, the complex 530 including one of the firsttype of functionalized bead 110, the analyte 120, and one of the secondtype of functionalized bead 130, and a microwave source 538 configuredto generate a microwave field incident on the at least one ODMR center540, the microwave source 538 being further configured to generate themicrowave field with frequencies that correspond to ground statetransitions in the at least one ODMR center 540, in which the at leastone ODMR center 540 produces emitted light 542 when illuminated by theincident light 536, characteristics of the emitted light 542 beinginfluenced by the microwave field and by the magnetic functionalizedbead 110 associated with the analyte 120 in the complex 330. In theembodiment shown in FIG. 5 , the plurality of ODMR centers 540 arenitrogen-vacancy (NV) centers in a diamond lattice, formed in an uppersurface of the diamond substrate 532. In another aspect, the pluralityof ODMR centers can be silicon-vacancy centers in a silicon carbidelattice, or in a diamond lattice. Turning back to FIG. 5 , under opticalexcitation 536, fluorescence 542 emitted from a thin layer of ODMRcenters 540 near the surface of the diamond substrate 532 is imaged ontoan optical photodetector array 544, that is an optical imaging systemhaving an imaging sensor such as a charge-coupled device (CCD) orcomplementary metal oxide semiconductor (CMOS) camera. The variation ofODMR center fluorescence under microwave excitation reveals the ODMRelectron spin resonance (ESR) frequency, and hence the magnetic fieldshift of the ODMR spin sublevels. The spatial structure of the magneticfield at the diamond surface created by the sample (i.e., complex) 530can thus be determined from images of ODMR center fluorescence 542,whose characteristics are influenced by the microwave field and by themagnetic field created by the magnetic functionalized bead 110associated with the analyte 120 in the complex 530.

Briefly, the process to acquire a magnetic image is as follows:

-   -   1. Dispose a magnetic sample (i.e., complex) 530 to be imaged        over, onto, or near to the sensing surface of the diamond        substrate 532. An intermediate layer (not shown) may be        interposed between the sample 530 and the diamond substrate 532.    -   2. Apply a magnetic bias field 534 in an arbitrary direction.    -   3. Illuminate the ODMR centers 540 in the diamond center with        green light 536 (near 532 nm wavelength).    -   4. Apply a microwave field from a source 538 to the diamond,        with frequency near one of the ODMR center ESR transitions.    -   5. Acquire an image of ODMR center fluorescence 542 emitted from        the sensing surface 540 at optical detector array 544 through        imaging objective 546 and optical filter 548.    -   6. Repeat steps 4-5 using different microwave frequencies that        span one or more ranges around one or more NV center ESR        transitions. The result is a stack of images, each corresponding        to a different microwave frequency.    -   7. Repeat steps 4-6 one or more times, averaging the results to        reduce imaging noise in the image stack.    -   8. For each image pixel in the image stack, construct an ESR        spectrum from that pixel's value across all images in the stack.        Analyze this spectrum to determine the frequencies of one or        more ESR transitions.    -   9. For each image pixel in the image stack, compute the magnetic        field based on the frequencies of observed ESR transitions at        that pixel.

Additional details of the operation of the wide-field diamond magneticimaging apparatus are described in PCT Patent Application No.PCT/US2017/057628 filed on Oct. 20, 2017 and entitled METHODS ANDAPPARATUS FOR MAGNETIC PARTICLE ANALYSIS USING DIAMOND MAGNETIC IMAGINGthat is incorporated by reference herein.

An applied magnetic field induces magnetization in bead A and anassociated magnetic field from the bead. A magnetic field in the rangeof 0.5 to 10 mT, which may be generated with permanent magnets or anelectromagnet, is sufficient to resolve features in the electron spinresonance spectrum of the diamond imaging sensor. The diamond magneticimager images these bead fields directly, allowing for individual beaddetection and location. Beads of similar composition and magnetizationproduce similar magnetic field patterns that may be identified ascharacteristic features 542 in a magnetic image corresponding to thelocation of each bead A. A representative image is shown in FIG. 6C.

An image processing algorithm may identify the locations both of bead Afeatures 542 in the magnetic image, shown in FIG. 6C, and bead Bfeatures 155 in the fluorescence image shown in FIG. 6B. Additionalimages may be acquired in either detection channel (magnetic orfluorescent) to improve signal fidelity. The resulting bead locationsidentified in each detection channel may then be compared to identifyco-presence of both bead types, and hence of complexes 145 containingthe target analyte, illustrated in FIG. 6A.

Adding the magnetic imaging channel to detect bead A in addition todetecting bead B in the bead fluorescence channel allows foridentification of unbound bead B, which may persist after magneticseparation or which may dissociate from bead complexes that are weaklybound by nonspecific interactions. Unbound bead B may be rejected duringanalysis so that only bead B associated with complexes are counted.

Example D4: Fully Magnetic Assay with Magnetic Imaging

In another embodiment shown in FIGS. 7A-7D, bead A 710 and bead B 730are both magnetic, but with distinguishable magnetic properties.Magnetic imaging with single-bead spatial resolution is used to identifybead A 710, as in Example C3, and also to identify bead B 730,distinguishing between the two. Bead A 710 has magnetic propertiessuitable for magnetic separation, as in Examples A1, B2, and C3described above.

Beads A 710 and B 730 may, for example, differ in the shape andmagnitude of their single-axis magnetization curves, which describe beadmagnetization as a function of an applied magnetizing field. Beads A 710and B 730 may differ in the degree of hysteresis in their magnetizationcurves and in properties such as remanent magnetization and coercivity.Beads A 710 and B 730 may have different degrees of asymmetry, withdifferent magnetization curves observed when the field axis is changed.Beads A 710 and B 730 may respond differently to a time-varying magneticfield, such as an alternating or rotating field.

Using only magnetic imaging for identifying and locating bead A 710,bead B 730, and complexes 745 including the analyte 720 enableselimination of the optical fluorescence detection channel, simplifyingthe assay system significantly. Additionally, magnetic imaging isparticularly insensitive to signal backgrounds due to unwanted light,detector noise, and sample contaminants that fluoresce, scatter, orabsorb light. Magnetic signal backgrounds are extremely low inbiological samples and they do not impede the ability to measure evenmodestly magnetic beads.

Distinguishing Magnetic Bead Types with Magnetic Imaging

Wide-field diamond magnetic imaging provides a means to directly imagethe vector magnetic field produced by a magnetic bead under a wide rangeof magnetic conditions. This general-purpose tool may be used todistinguish between magnetic bead types over a wide range of differentproperties.

In one embodiment, bead A 710 and bead B 730 are distinguished bymeasuring magnetic susceptibility and magnetic remanence at low appliedfield after first magnetizing the beads with a large magnetic field.Bead A 710 is superparamagnetic. For example, bead A 710 may be composedof superparamagnetic iron oxide nanoparticles 5-10 nm in size dispersedwithin a spherical polymer substrate approximately 1 μm in diameter.Bead A 710 may contain a quantity of iron oxide such that the magnitudeof the average induced magnetization of bead A 710 with an applied biasfield of 4 mT is approximately 3×10⁻¹⁵ A m². Bead B 730 isferromagnetic. In one embodiment, bead B 730 may be composed offerromagnetic cobalt ferrite nanoparticles 30 nm in size dispersed overthe surface of a spherical polymer substrate approximately 1 μm indiameter and adhered to the surface with an additional polymer layer.Bead B 730 has a remanent magnetization fraction of greater than 50%,such that, after being magnetized in a field of at least 300 mT and oncethe magnetizing field has been removed, bead B 730 retains a largeproportion of its saturated magnetization value. Bead B 730 may containa quantity of cobalt ferrite such that the magnitude of the averageremanent magnetization of bead B 730 after the magnetizing field isremoved is approximately 2×10⁻¹⁵ A m².

A magnetic imaging procedure is described below for identifyingcomplexes 745 containing the target analyte 720, bead A 710 and bead B730.

After forming complexes 745 in a sample suspension, a representativeportion of the sample is disposed over and dried on the surface 732,shown in FIG. 7B, of a diamond magnetic imaging sensor shown in FIG. 5 .The sensor's imaging surface is a {100} face and this surface contains athin layer approximately 1-μm thick that is rich in nitrogen-vacancy(NV) centers. Turning back to FIGS. 7B-7D, after magnetic imaging,complexes 745 are identified by identifying bead A 710 and bead B 730 inclose proximity to one another, including close enough to be spatiallyunresolved in the images. Prior to magnetic imaging, a magnetizing fieldis applied in a direction normal to the horizontal diamond surface. Afield of greater than 200 mT applied for a period of several seconds issufficient to magnetize the magnetic material in bead B. The driedsample is then magnetically imaged twice with a bias magnetic field of 4mT applied parallel to one crystal axis of the diamond sensor, which isoriented at an angle of approximately 35 degrees with respect to theimaging surface. The 4 mT imaging field is reversed between acquiringthe two magnetic images, shown in FIGS. 7C and 7D, termed the positive(FIG. 7C) and negative (FIG. 7D) images, denoting the +4 mT and −4 mTimaging fields, respectively. The magnetic images measure the projectionof the sample magnetic field vector onto the axis of the imaging field.

Since bead A 710 is superparamagnetic, the greater than 200 mTmagnetizing field does not leave bead A 710 with significant remanentmagnetization. In both the positive and negative images, themagnetization of bead A is only that which is induced in thesuperparamagnetic beads by the 4 mT imaging field. Bead A 710 producesthe same feature 741 in both magnetic images, since the bead A 710magnetization is in both cases parallel to the imaging field.

In contrast, the greater than 200 mT magnetizing field leaves bead B 730strongly magnetized in the vertical direction, oriented up with respectto the horizontal diamond sensor imaging surface. Once the magnetizingfield is removed, the weaker 4 mT imaging field does not significantlychange the magnetization of bead B 730, since the magneticsusceptibility of bead B 730 near zero magnetic field, when previouslymagnetized along the same axis, is low. Therefore, bead B 730 producesan image feature 742 that inverts sign between the positive and negativemagnetic images, with positive magnetic field projection changing tonegative and vice versa, as illustrated in FIGS. 7B, 7C, and 7D.

All magnetic objects identified in the magnetic image field of view arequantified by magnetization, such that bead A 710 is assigned a positivevalue in both images and bead B 730 is assigned a positive and negativevalue in the positive and negative images, respectively. Bead complexes745 will be assigned magnetization values that reflect the complexcomposition. For example, bead dimers of the form A-A or B-B willgenerally be assigned larger values with the same sign of bead A or beadB monomers, respectively. Bead dimers 745 of the form A-B or largerheterogeneous bead complexes will be assigned values of smallermagnitude in the negative image than in the positive image, reflectingoppositely-magnetized beads within the complex, as shown in FIGS. 7C and7D.

All magnetic objects in the magnetic images may be represented on ascatter plot whose axes are the sum and difference, respectively, of thepositive and negative image magnetization values. This sum anddifference may also be termed the susceptibility and remanence of thesingle-bead magnetization curve, as they are approximately proportionalto these properties. As shown in FIG. 8A, bead A and bead complexescontaining only bead A will be clustered near one axis, with largesusceptibility and zero remanence; bead B and bead complexes containingonly bead B will be clustered near the other axis, with large remanenceand near-zero susceptibility. Complexes containing both bead A and beadB will exhibit significant susceptibility and remanence, so they may beidentified as the objects in the scatter plot in a region sufficientlyseparated from both axes. This region is unlikely to contain signalsfrom bead A or bead B alone, or from homogeneous bead complexes such asthose of the form A-A or B-B.

If the magnetic imaging spatial resolution is sufficient to resolveindividual magnetic beads within a complex, then the complex may beidentified by separately identifying beads within the complex anddetermining their spatial separation to be consistent with that of abound complex, and not significantly greater than the bead diameters.

If both bead A and bead B are sufficiently magnetic, and either bead Aor bead B is ferromagnetic, A-B dimers may form even in the absence ofthe target analyte, due to attractive magnetic interactions. Thesemagnetic interactions may be limited in strength by limiting the amountof magnetic material in each bead. Magnetic bead signals may be measuredeven in cases in which magnetic interactions between beads are too weakto overcome forces associated with Brownian motion or sample mixing, sothat magnetic interactions may play no role.

The magnetic material within bead A and bead B may be composed ofnanoparticles disposed within or on the surface of a polymer or othernonmagnetic substrate. If the nanoparticles are uniformly disposedwithin or on the surface of the substrate, then the strength of magneticinteractions between beads can be reduced relative to having aggregatednanoparticles, since magnetic fields near aggregated magneticnanoparticles may be stronger. Using nanoparticles that are much smallerthan the substrate radius may allow for more uniform distribution,relative to larger nanoparticles that produce stronger local magneticfields.

Magnetic interactions may also be suppressed by adding a nonmagneticlayer encapsulating the magnetic material. Suitable materials for thenonmagnetic layer include polymers, such as polyethylene (PE),polytetrafluoroethylene (PTFE), and polymethylmethacrylate (PMMA). Sincemagnetic interactions weaken rapidly with increasing separation betweenbeads, even a nonmagnetic layer significantly thinner than the originalbead radius can dramatically reduce dimer formation due to magneticinteractions.

Additional Magnetic Discrimination Methods Discrimination by MagneticMoment

As shown in FIG. 8B, the magnetic image signal for a magnetic bead canbe analyzed to determine the magnetic moment (magnetization×volume) ofthe bead, assuming knowledge of the bead size and a sphericallysymmetric distribution of magnetic material in the bead. For bead A andbead B of similar size, the magnetic moment can be used to distinguishbetween bead A, bead B, and bead complexes. To be effective, there mustbe low enough variation of magnetic moment, size, and spherical symmetryof each bead such that each measurement can be clearly associated withone distribution. The A-B complex, having larger size than eachindividual bead, may not produce a signal equal to the sum of signalsfrom bead A and bead B. Nevertheless, the bead A and bead B magneticmoments may still be chosen such that the mean A-B complex signal isdistinct from that of bead A, bead B, the A-A complex, the B-B complex,etc. It is not necessary to resolve spatial differences betweencandidate signals to discriminate them by magnetic moment; it issufficient to evaluate each signal only by magnitude, e.g. magnitude ofconvolution with a characteristic image signal.

If bead A and B have different size, a similar discrimination approachmay be used that ignores this size difference when evaluating themagnitude of candidate signals and applies the same single-parameterquantification strategy to all signals. This may produce signals forbead A, bead B and complexes that are not proportional to their magneticmoments, but are distinct and allow for accurate discrimination.

Discrimination by Anisotropy

Magnetic particles may exhibit an anisotropic response to a magneticfield, due to preferential magnetization along certain crystal axes in asingle magnetic domain or along certain directions in a multi-domainparticle or a composite magnetic bead containing many particles.Rod-shaped nanoparticles, for example, typically can be magnetized moreeasily along the rod axis. Synthesizing a spherical bead containingoriented magnetic nanorods would produce an anisotropic magneticsusceptibility in the bead.

The magnetic anisotropy of a bead can be probed by imaging immobilizedbeads multiple times, using multiple directions of an applied magneticfield. As shown in FIG. 8C, a metric for magnetic anisotropy can beconstructed from the difference in magnetic signals obtained from thedifferent orientations. Imaging at three distinct directions issufficient to determine the orientation and degree of anisotropy for aparticle even if the particle orientation is not known in advance. Ifbead A and bead B have zero and nonzero magnetic anisotropy,respectively, then images acquired with the imaging magnetic fieldrotated in different directions will produce identical signals for beadA, but different signals for bead B. Complex signals will have nonzeroanisotropy, but less than that of bead B.

Discrimination by Coercivity

As shown in FIG. 8D, magnetized ferromagnetic beads can be re-magnetizedin a different direction by applying a field larger than the coercivity.Discrimination between two types of ferromagnetic beads, bead A and beadB, can be achieved using this sequence: (1) first magnetize both beadswith a strong magnetic field in one direction; (2) image the beadmagnetization; (3) apply a magnetic field in the opposite direction thatis strong enough to reverse the magnetization of bead A, but not strongenough to reverse the magnetization of bead B; (4) image the magneticbead signals and compare them to those in the first image. Bead Asignals will reverse direction; bead B signals will change modestly, ifat all; complex signals will change significantly in magnitude as onebead in the complex reverses magnetization while the other does not.

Discrimination by Time-Dependent Magnetic Response

As shown in FIG. 8E, magnetic particles change their magnetizationdirection in response to a change in magnetic field direction. For agiven field strength, the time scale for a particle to change directionmay depend on the particle composition and size and may vary over a widerange from below 1 μs to well over 1 s. If an oscillating or rotating ACmagnetic field of constant amplitude is applied to the particle, theparticle magnetization will oscillate in response. An oscillatingmagnetization may be measured by a magnetic imaging technology that issensitive to AC magnetic fields, such as a wide-field ODMR centermagnetic imaging system that employs pulsed optical excitation of ODMRcenters or time-gated camera exposures. The magnitude of the oscillatingmagnetization will decrease as the oscillation period decreases belowthe time scale required for the particle to change magnetizationdirection. The cutoff frequency is defined as the oscillation frequencycorresponding to this change in response.

If bead A and bead B contain magnetic material with different cutofffrequencies, measuring the oscillating magnetization at multipleoscillation frequencies provides a method to discriminate between thebeads. If bead A has a high cutoff frequency compared to bead B, thenimaging at an intermediate frequency will observe a weak bead B signalcompared to imaging at a low frequency, but little change in the bead Asignal. A complex will exhibit a decrease in signal at the intermediatefrequency that is smaller than that of bead B. Discrimination can beimproved by adding additional images at additional frequencies. Whilebead A and bead B will have a single cutoff frequency, the complex willexhibit two cutoff frequencies. Signals obtained at low oscillationfrequency and at two or more intermediate frequencies will revealqualitatively different behavior for bead A, bead B, and complexes.

Discrimination by Magnetic Saturation

As shown in FIG. 8F, the magnetization M of a superparamagnetic particlesaturates with sufficiently high magnetic field H. Even at fieldstrengths below saturation, the magnetic susceptibility (slope of themagnetization curve) is reduced. If the magnetizations ofsuperparamagnetic bead A and bead B saturate at different fieldstrengths H₁ and H₂, then the beads may be distinguished by imaging attwo magnetic field strengths, one of which is large enough to observe achange in magnetic susceptibility in one of the beads. The ratio ofsignals in these two images will be significantly different for bead Aand bead B. Complexes will have an intermediate ratio distinct from thatof bead A or bead B.

Size Based Magnetic Bead Discrimination

Magnetic beads of different size, but similar composition, may producemagnetic image signals that are distinguishable by their spatial scale.This may allow for discrimination between bead A, bead B, and complexes,despite bead A and bead B having nominally identical magneticproperties.

When the sample solution is disposed on a surface, most beads will cometo rest against the surface, so that the center of each bead is spacedfrom the surface by its radius. Larger beads are thus centered furtherfrom the sensing surface than smaller beads. This spacing determines thespatial scale of the magnetic field at the sensing surface, since thesame lateral displacements along the surface are relatively larger forclosely-spaced beads than for more distant beads, and therefore resultin larger relative changes in magnetic field.

FIG. 9 is an image of two beads, approximately 1 micron (top) and 3microns (bottom) in diameter, with their centers spaced approximately 9microns apart. The larger bead produces a magnetic signal with broaderspatial features. In this case, the larger bead also contains moremagnetic material and produces a larger magnitude of signal, but thisneed not be the case.

If bead A and bead B have different size, but similar magneticproperties, then spatial scale of magnetic image signals may be used notonly to discriminate between the two, but also to identify A-Bcomplexes. Complexes have spatially broad signals that also containshorter-scale spatial components.

One method for identifying complexes is to first identify all broadsignals (including both bead B and complex signals) and then subtract acharacteristic bead B signal (such as the mean of many bead B signalsimaged separately) from each. Variations in imaging accuracy and in theuniformity of bead B magnetization will cause this difference to benonzero for bead B signals, however the difference will generally havebroad spatial scale. For the complex signals, however, subtracting thecharacteristic bead B signal will leave behind the sharper bead Asignal. These cases may be distinguished by spatial filtering of thesignal differences.

FIGS. 10A-1, 10B-1, and 10C-1 show example bead B images. The differencesignal images after subtracting the characteristic bead B signal shownin 10A-2, 10B-2, and 10C-2 have a gray scale amplified by a factor of 2.

FIGS. 11A-1, 11B-1, and 11C-1 show example complex images, showing sharpbead A signals circled in the difference images shown in 11A-2, 11B-2,and 11C-2, which are again amplified by a factor of 2 relative to theimage signals.

Combined Approaches

The bead discrimination approaches described herein may also be used incombination to enhance discrimination performance or to discriminatebetween more than two bead types and their combinations.

Additional Assay Features Accounting for Variation in Bead Density

For a given number of bead complexes containing the target analyte in aliquid sample suspension, the number of complexes present within animaging field of view after disposing the sample over the imaging sensormay vary due to differences in the manner in which the sample wasdisposed. For example, the sample may be disposed over the sensor byadding a liquid droplet to the sensor surface and allowing it to dry,such that variations in the droplet volume or its initial contact areawith the sensor lead to variations in bead complex density in the driedsample over the sensor surface. By measuring the total number of bead Ain the field of view, including the unbound beads not contained incomplexes, the sample density variations may be measured and accountedfor. Dividing the number of complexes by the number of bead A yields aquantity that is less sensitive to variations in sample density, andthus may provide a more precise measurement of the total number of beadcomplexes in the sample and of the analyte concentration determined froma calibration curve obtained as described above.

Accelerated Bead Interaction Kinetics

It is known in the art that immunoassays must allow time for targetanalytes in a liquid sample to bind to antibodies that enable detectionof the target analytes. Depending on the reagent concentration andsample conditions (such as temperature, viscosity, and process foragitating or mixing the sample), several minutes may be required formost analytes to become bound, even when there is a large excess ofbinding sites available, due to the time needed for the analyte to movethrough the sample by diffusion or active shaking or stirring.

The rate of interactions between different beads in the samplesuspension may determine multi-bead assay speed, since bead diffusion isgenerally slower than diffusion of smaller molecular analytes. Since abead-bound target analyte may also occupy a relatively small fraction ofthe bead's surface area, when the bead to which the analyte is boundinteracts with a second bead, the analyte may not be exposed to thesecond bead in a manner conducive for binding (e.g., the interactionoccurs on the side of the first bead opposite to where the analyte islocated). Several bead interactions may be required on average to forman immunocomplex. The multi-bead assay time may be shortened byperforming processes to induce bead-bead interactions that lead toimmunocomplex formation, accelerating bead kinetics beyond what may beexpected for diffusion or stirring alone.

In one embodiment, bead-bead interactions may be induced byagglomerating a plurality of functionalized beads of the first andsecond types, after contacting the sample solution with the populationof functionalized beads of the second type, before detecting thecomplex, by means of spinning the sample suspension on a centrifuge toconcentrate the beads into a pellet in the sample tube. This process canbe performed in less than a minute with standard benchtop centrifugesystems. Beads in the pellet may be closely spaced or in contact withone another, resulting in many bead interactions in the pellet. Thepellet may be re-suspended by mixing the suspension. This centrifugeprocess may be repeated as necessary to ensure sufficient interactionsbetween beads to form immunocomplexes containing the target analyte.

In another embodiment, if both bead A and bead B are magnetic, thenmagnetic separation may be used to form a pellet of beads in the samplesuspension and induce bead-bead interactions by applying a magneticfield gradient to the sample solution after contacting the samplesolution with the functionalized beads of the first and second types. Aswith the centrifuge process, this magnetic approach to accelerating beadkinetics can be performed in less than a minute and repeated asnecessary to form immunocomplexes containing the target analyte. Themagnetic approach may be performed with permanent magnets for a simple,inexpensive, and compact process with minimal power consumption. Anelectromagnet may also be used to apply the magnetic field with nomoving parts.

In another embodiment, a bead pellet produced by magnetic separation ofa sample suspension can be agitated without removing the magnetic field,but by varying or otherwise changing the magnetic field gradient appliedto the sample solution with respect to the beads. For example, the fieldmagnitude, direction, or spatial distribution may be changed oroscillated to apply different magnetic forces on the beads.Alternatively, or additionally, the sample tube may be moved withrespect to the magnetic field. For example, rotating the tube may movethe pellet away from its equilibrium position so that the pellet will bedragged by the magnetic field gradient to a new position. These changeswill cause beads in the pellet to move with respect to each other andmay induce additional bead interactions and immunocomplex formation.

In an embodiment, a permanent magnet may be moved relative to a tubecontaining a sample suspension with magnetic beads of multiple types.The magnet may follow a fixed pattern of motion. Exemplary cases includethe magnet orbiting the sample tube in a circle, rotating on its ownaxis, or rocking back and forth between two points. The motion may becontinuous, in which case the bead pellet will continuously move throughthe tube, subjecting the beads to shear forces from the liquid, tubewalls, and other beads. This motion and the associated forces on thebeads will agitate the pellet continuously to drive bead-beadinteractions. In another exemplary case, the motion may occur indiscrete periods separated by periods of rest, in which the bead pelletmay concentrate to a higher bead density than is achieved duringcontinuous motion. If the different bead types respond significantlydifferently to the field of the permanent magnet, then the periods ofrest will allow the multiple bead types to co-localize more effectivelythan during continuous motion.

In another embodiment, a plurality of permanent magnets may be movedrelative to a plurality of samples in separate wells of a plate, suchthat the sample in each well is subjected to a magnetic field profile intime and space that is substantially similar. This approach allows fordriving bead-bead interactions in parallel over a plurality of samplesfor improved sample preparation throughput.

Accelerating bead kinetics and the rate of bead-bead interactions insample suspension decreases the time required to bind target analytesinto detectable multi-bead complexes. This method enables a rapid assay.

This method may also allow for a lower quantity of binding ligands to beused on the bead surfaces, since the likelihood of a given ligand tobind to the target analyte may be increased by the increased frequencyof bead-bead interactions. Using fewer binding ligands may reduce thecost of the assay significantly.

Multiplexing

It is often useful for an assay to measure concentration of multipledistinct analytes in a single sample. A multiplexed assay measuresdistinct target analytes by associating a distinct signal with eachtarget, so that the analyte signals may be distinguished in the assaymeasurement. The magnetic dual-bead assay may be generalized to amultiplexed multi-bead assay by using more than two distinguishable beadtypes. Different analytes may be specifically detected by observing theformation of analyte-specific complexes including a plurality offunctionalized beads of at least a third type, functionalized to includeat least a third moiety that can specifically associate with at least asecond analyte under appropriate conditions.

In one embodiment, shown in FIGS. 12A and 12B, consider a plurality offunctionalized beads of the third type 1240 functionalized to include atleast a third moiety 1245 that associates with the second analyte 1250under suitable conditions, and a plurality of functionalized beads of afourth type 1260, functionalized to include a fourth moiety 1265 thatassociates with the second analyte 1250 under suitable conditions,resulting in four distinguishable bead types: bead A 1210, bead B 1230,bead C 1240, and bead D 1260. Complexes of the form A-B 1201 or C-D 1202may be formed through binding of two distinct analytes, first analyte X1220 and second analyte Y 1250, to moieties coating each bead. In thiscase, bead A 1210 and bead B 1230 are coated with moieties 1215 and1235, respectively, targeting two distinct regions X1 and X2 of analyteX 1220; bead C 1240 and bead D 1260 are coated with moieties 1245 and1265, respectively, targeting two distinct regions Y1 and Y2 of analyteY 1250. The moieties on each bead are unique to that bead. The A-B 1201and C-D 1202 complexes may be distinguished from each other and from themonomer beads from their distinct magnetic properties.

In another embodiment, shown in FIGS. 13A and 13B, consider threedistinguishable bead types: bead A 1310, bead B 1320, and bead C 1330.Complexes of the form A-B 1301, B-C 1302, or A-C 1303 may be formedthrough binding of three distinct analytes, analyte X 1311, analyte Y1312, and analyte Z 1313, to moieties coating each bead. Thefunctionalized beads of the first type A 1310 further include at leastone additional moiety 1316 that associates with the second analyte 1312under suitable conditions. The functionalized beads of the second type B1320 further include a moiety 1326 that associates with the thirdanalyte Z 1313 under suitable conditions. The functionalized beads ofthe third type C 1330 are functionalized to include a moiety 1335 and amoiety 1336, and therefore each bead is coated with two distinct moietytypes targeting two different analytes: bead A 1310 is coated withmoieties for analyte X 1315 and for analyte Y 1316; bead B 1320 iscoated with moieties for analyte X 1325 and for analyte Z 1326; bead C1330 is coated with moieties for analyte Y 1335 and for analyte Z 1336.The moieties on each bead are unique to that bead. The A-B 1301, B-C1302, and A-C 1303 complexes may be distinguished from each other andfrom the monomer beads from their distinct magnetic properties.

Multiplexed assays must have a means of discriminating between signalsassociated with distinct target analytes, which in the case ofmultiplexed bead assays means that the beads must be distinguishable.Bead fluorescence may be used to discriminate between bead types withdifferent excitation and/or emission spectra. For example, three beadtypes may emit blue, yellow, or red fluorescence that may bedistinguished using optical filters. Different bead types may also beprepared to fluoresce with different intensities, so that the differenttypes may be distinguished in a fluorescence image by discrete levels ofbrightness, where difference between the levels exceeds variation withinthe distribution of each bead type.

Distinct magnetic bead types may be distinguished by preparing beadtypes with different magnetic properties that can be distinguished bymagnetic imaging. In an exemplary case, the distinct bead types may beprepared by loading each bead type with specific and distinguishablequantities of magnetic material. In another exemplary case, distinctbead types may be prepared by loading each bead type with differentmagnetic material exhibiting different properties. The fully magneticmulti-bead assay described above discriminates between two bead types inthis manner. A multiplexed assay may be implemented by adding additionaldistinguishable beads using distinct combinations of the propertiesdescribed above. For example, bead A may be superparamagnetic, whilebead B and C are ferromagnetic with different coercivities derived frombead B and bead C comprising different ferromagnetic materials. In thiscase, the beads may be distinguished by measuring magnetic remanenceafter magnetization, and then also measuring whether this remanence isreversed upon application of a demagnetizing field that exceeds thecoercivity of bead B, but not that of bead C. The three beads in thiscase may be used to implement a multiplexed assay for three analytesusing an embodiment described above.

Sample Preparation

One example of suitable conditions for sample preparation for themulti-bead assay is to combine a few drops of blood with the multi-beadmixture, incubate for a few minutes with accelerated kinetic mixing anddeposit the sample solution on the diamond surface to dry followed bymagnetic imaging. The sample solution can be partially or completelydehydrated before detecting the complex.

A suitable sample preparation proceeds as follows: plasma or serum isdiluted in assay buffer 10-fold by adding 5 μL sample into 45 μL assaybuffer and briefly vortex mixed. This diluted sample is further diluted2× with 50 μL of bead mix for a final of 100 μL. The bead mix includes˜100,000 capture beads and ˜100,000 detector beads. The final assayreaction is 20-fold dilution of sample in 100 μL. The assay reaction isincubated with vortex mixing (800 rpm) for 15 minutes at roomtemperature. The samples are then placed in a centrifuge and spun at1500 g for 3 minutes, followed by pulse vortex mixing. Thecentrifugation and mixing cycle is repeated twice more, after which thesample is placed against a permanent magnet (magnetic field ˜300 mT) for30 seconds to pellet the magnetic beads against the sidewall of thereaction tube. The assay volume is removed by pipette leaving the beadpellet intact on the side wall against the magnet. The tube is removedfrom the magnet and the pellet is suspended in 500 μL of wash buffer byvortex mixing. The tube is pulse spun at 1500 g for 3 seconds to removefluid from the cap, and placed on the magnet for 30 seconds. The washcycle is repeated 2 more times for a total of 3 washes. The pellet iswashed 1 time with 200 μL of imaging buffer and finally suspended in ˜4μL imaging buffer. ˜2 μL is applied to the diamond sensor for magneticimaging.

The sample may be any chemical or biological sample, such as wholeblood, blood components (plasma, serum), tissue culture, cell culture,bodily fluids (cerebral spinal fluid (CSF), tears, saliva, breast milk,urine, semen, nasal discharge), tissue samples (oral swabs, biopsies,surgical resections), recombinant DNA, RNA or protein, endogenous DNA,RNA or protein, synthetic nucleic acids or protein peptides.

Further sample requirements may include volumes of sample types from 0.1μL to 1000 μL.

Further sample requirements may include dilution of sample types andvolume into assay buffers. Dilutions of sample types may includedilution by a factor of 10-1,000. Assay buffers may be determinedempirically for optimized signal generation and minimized non-specificbackground, or false binding of any kind.

Samples may be combined in various ways including, for example, withmulti-bead mixtures in blood collection tubes, assay tubes, assayplates/well, microfluidic devices, reaction chambers, incubationchambers, lateral flow devices, blood component separation devices, orother liquid handling or manipulation devices.

Samples may be mixed in various ways including, for example, by magneticfields, centrifugal force, gravity, sound induced, light induced,electric induced, ionic interactions, van der Waals induced, Brownianmotion, spinning, or other mechanical means.

Samples may be mixed with multi-bead mixtures for times necessary tocapture targets of interest ranging from, for example, a second toseveral hours.

Samples may be introduced to the magnetic imaging device in various waysincluding, for example, by pipette, capillary flow tube or device,sample handling device, liquid handling device, integrated device,lateral flow device, disposable or reusable device.

Samples may be deposited on the diamond surface by several modes ofapplication including, for example, pipetting, pouring, dripping,capillary flow, pumping, gravity induced flow, magnetic induced flow,ionic induced flow, sound induced flow, light induced flow, mechanicalvibration induced flow, sheath flow, centrifuge induced, and thermalinduced flow.

Samples may be magnetically imaged in a dry, dehydrated (i.e., partiallydry or gel), or wet state.

FURTHER EXAMPLE EMBODIMENTS

Example 1 is a bead-based magnetic assay system for detecting a complexincluding an analyte based on optically detected magnetic resonance(ODMR), the system comprising: (a) a plurality of functionalized beadsof a first type, which are magnetic functionalized beads and arefunctionalized to include a first moiety that associates with an analyteunder suitable conditions, (b) a plurality of functionalized beads of asecond type, which are functionalized to include a second moiety thatassociates with the analyte under suitable conditions, (c) a substrateincluding at least one ODMR center, (d) a light source configured togenerate incident light that excites electrons within the at least oneODMR center from a ground state to an excited state, (e) a magnet forapplying a bias magnetic field on a complex disposed over the at leastone ODMR center, the complex including one of the first type offunctionalized bead, the analyte, and one of the second type offunctionalized bead, (f) a microwave source configured to generate amicrowave field incident on the at least one ODMR center, the microwavesource being further configured to generate the microwave field withfrequencies that correspond to ground state transitions in the at leastone ODMR center, in which the at least one ODMR center produces emittedlight when illuminated by the incident light, characteristics of theemitted light being influenced by the microwave field and by themagnetic functionalized bead associated with the analyte in the complex,and an optical photodetector that detects light emitted by the at leastone ODMR center.

Example 2 includes the subject matter of Example 1, wherein the at leastone ODMR center is a silicon vacancy center in a silicon carbidelattice.

Example 3 includes the subject matter of Example 1, wherein the at leastone ODMR center is a silicon vacancy center in a diamond lattice.

Example 4 includes the subject matter of Example 1, wherein the at leastone ODMR center is a nitrogen-vacancy center in a diamond lattice.

Example 5 includes the subject matter of Example 4, wherein the at leastone ODMR center is formed in an upper surface of the substrate.

Example 6 includes the subject matter of Example 5, wherein the at leastone ODMR center is a plurality of ODMR centers formed in the uppersurface of the diamond substrate.

Example 7 includes the subject matter of Example 6, wherein the opticalphotodetector is an optical imaging system having an imaging sensor thatimages the emitted light from the plurality of ODMR centers.

Example 8 includes the subject matter of any of Examples 1-7, whereineach of the first and the second moiety is a receptor, protein,antibody, cell, virus, or nucleic acid sequence.

Example 9 includes the subject matter of any of Examples 1-8, whereinthe functionalized beads of the first type are superparamagneticfunctionalized beads including a superparamagnetic material.

Example 10 includes the subject matter of Example 9, wherein thefunctionalized beads of the first type include a nonmagnetic layerencapsulating the superparamagnetic material.

Example 11 includes the subject matter of Example 9, wherein thesuperparamagnetic functionalized beads include iron oxide particles.

Example 12 includes the subject matter of any of Examples 1-11, whereinthe functionalized beads of the first type comprise magneticnanoparticles disposed within a polymer substrate.

Example 13 includes the subject matter of any of Examples 1-11, whereinthe functionalized beads of the first type comprise magneticnanoparticles disposed on a surface of a polymer substrate.

Example 14 includes the subject matter of any of Examples 1-13, whereinthe functionalized beads of the second type are fluorescentfunctionalized beads.

Example 15 includes the subject matter of any of Examples 1-13, whereinthe functionalized beads of the second type are magnetic functionalizedbeads including a quantity of magnetic material distinguishable from thefunctionalized beads of the first type.

Example 16 includes the subject matter of any of Examples 1-13, whereinthe functionalized beads of the second type are magnetic functionalizedbeads, the second type of functionalized beads including a magneticproperty distinguishable from the functionalized beads of the firsttype.

Example 17 includes the subject matter of Example 16, wherein thefunctionalized beads of the first type are superparamagneticfunctionalized beads including a superparamagnetic material.

Example 18 includes the subject matter of Example 17, wherein thefunctionalized beads of the first type include a nonmagnetic layerencapsulating the superparamagnetic material.

Example 19 includes the subject matter of Example 16, wherein thefunctionalized beads of the second type are ferromagnetic functionalizedbeads including a ferromagnetic material.

Example 20 includes the subject matter of Example 19, wherein thefunctionalized beads of the second type include a nonmagnetic layerencapsulating the ferromagnetic material.

Example 21 includes the subject matter of any of Examples 1-20, whereineach of the first type of functionalized beads and the second type offunctionalized beads has a diameter in a range of between 50 nm and 10μm.

Example 22 includes the subject matter of Example 21, wherein each ofthe diameters of the functionalized beads of the first type and thesecond type is in a range of between 0.5 μm and 5 μm.

Example 23 includes the subject matter of Example 21, wherein thediameter of the functionalized beads of the first type is similar to thediameter of the functionalized beads of the second type.

Example 24 includes the subject matter of Example 21, wherein thediameter of the functionalized beads of the first type is different fromthe diameter of the functionalized beads of the second type by at least50%.

Example 25 includes the subject matter of any of Examples 1-24, furtherincluding a plurality of functionalized beads of at least a third type,functionalized to include at least the second moiety that canspecifically associate with at least a second analyte under appropriateconditions.

Example 26 includes the subject matter of Example 25, further includinga plurality of functionalized beads of a fourth type, functionalized toinclude the second moiety that associates with the second analyte undersuitable conditions.

Example 27 includes the subject matter of Example 25, wherein thefunctionalized beads of the first and/or second type further include atleast one additional moiety that associates with the second analyteunder suitable conditions.

Example 28 includes the subject matter of Example 27, further includinga third moiety that associates with a third analyte under suitableconditions, wherein the functionalized beads of the first type arefurther functionalized to include the second moiety, and thefunctionalized beads of the second type are further functionalized toinclude the third moiety.

Example 29 is a method of detecting a complex including an analyte, themethod comprising: (a) contacting a sample in a solution with apopulation of functionalized beads of a first type, which are magneticfunctionalized beads and are functionalized to include a first moietythat associates with an analyte under suitable conditions, (b)contacting the sample solution with a population of functionalized beadsof a second type, which are functionalized to include a second moietythat associates with the analyte under suitable conditions, contactresulting in formation of a complex including one of the first type offunctionalized bead, the analyte, and one of the second type offunctionalized bead; and (c) detecting the complex including the analyteby detecting magnetic fields produced by the magnetic functionalizedbead and by detecting the functionalized bead of the second typeassociated with the analyte in the complex.

Example 30 includes the subject matter of Example 29, further includingdisposing the sample solution including the complex over a substratethat includes at least one optically detected magnetic resonance (ODMR)center formed in the substrate; exciting electrons within the at leastone ODMR center from a ground state to an excited state with incidentlight; applying a bias magnetic field on the complex; and generating amicrowave field incident on the at least one ODMR center, the microwavefield including frequencies that correspond to ground state transitionsin the at least one ODMR center, wherein detecting the complex includingthe analyte further includes analyzing light emitted by the at least oneODMR center, characteristics of the emitted light being influenced bythe microwave field and by the magnetic functionalized bead associatedwith the analyte in the complex.

Example 31 includes the subject matter of Example 30, wherein the atleast one ODMR center is a nitrogen-vacancy center in a diamond lattice.

Example 32 includes the subject matter of Example 31, wherein the atleast one ODMR center is formed in an upper surface of the substrate.

Example 33 includes the subject matter of Example 32, wherein the atleast one ODMR center is a plurality of ODMR centers formed in the uppersurface of the substrate.

Example 34 includes the subject matter of Example 33, wherein analyzinglight emitted by the plurality of ODMR centers includes imaging theemitted light.

Example 35 includes the subject matter of any of Examples 29-34, furtherincluding applying a magnetic field gradient to the sample solutionafter contacting the sample with the population of functionalized beadsof the first type.

Example 36 includes the subject matter of Example 35, wherein applyingthe magnetic field gradient to the sample solution is performed aftercontacting the sample solution with the population of functionalizedbeads of the second type.

Example 37 includes the subject matter of any of Examples 29-36, whereinthe population of functionalized beads of the first type and thepopulation of functionalized beads of the second type are added to thesample solution sequentially.

Example 38 includes the subject matter of any of Examples 29-37, whereinthe functionalized beads of the second type are fluorescentfunctionalized beads, and the method further includes illuminating thecomplex with incident light that excites fluorescence within thefunctionalized beads of the second type and fluorescence imaging of thecomplex.

Example 39 includes the subject matter of any of Examples 29-37, whereinthe functionalized beads of the second type are magnetic functionalizedbeads, including a magnetic property distinguishable from thefunctionalized beads of the first type.

Example 40 includes the subject matter of any of Examples 29-39, furtherincluding applying a magnetic field gradient to the sample solutionafter contacting the sample solution with the functionalized beads ofthe first and second types.

Example 41 includes the subject matter of Example 40, further includingvarying the magnetic field gradient applied to the sample solution.

Example 42 includes the subject matter of any of Examples 29-41, furtherincluding concentrating the sample solution after contacting the samplesolution with the population of functionalized beads of the second type.

Example 43 includes the subject matter of any of Examples 29-42, furtherincluding agglomerating a plurality of functionalized beads of the firstand second types, after contacting the sample solution with thepopulation of functionalized beads of the second type, before detectingthe complex.

Example 44 includes the subject matter of any of Examples 29-43, furtherincluding dehydrating the sample solution after disposing the samplesolution over the diamond substrate.

Example 45 is a bead-based assay system for detecting a complexincluding an analyte, the system comprising: (a) a plurality offunctionalized beads of a first type, which are magnetic functionalizedbeads and are functionalized to include a first moiety that associateswith an analyte under suitable conditions, (b) a plurality offunctionalized beads of a second type, which are fluorescentfunctionalized beads, and are functionalized to include an unlabeledmoiety that associates with the analyte under suitable conditions, (c) alight source configured to generate incident light that excitesfluorescence within the functionalized beads of the second type, and (d)an optical photodetector that detects fluorescence emitted by thefunctionalized beads of the second type associated with the analyte in acomplex including one of the first type of functionalized bead, theanalyte, and one of the second type of functionalized bead.

Example 46 includes the subject matter of Example 45, wherein thefluorescent functionalized beads comprise a polymer substrateimpregnated with a fluorescent material.

Example 47 includes the subject matter of Example 45, wherein theoptical fluorescence detector includes a spectrophotometer.

Example 48 includes the subject matter of Example 45, wherein theoptical fluorescence detector includes an optical imaging sensor thatimages the fluorescence emitted by the functionalized beads of thesecond type associated with the analyte in the complex.

Example 49 includes the subject matter of any of Examples 45-48, whereinthe functionalized beads of the first type are superparamagneticfunctionalized beads.

Example 50 includes the subject matter of Example 49, wherein thesuperparamagnetic functionalized beads include iron oxide particles.

Example 51 includes the subject matter of any of Examples 45-50, whereinthe functionalized beads of the first type include magneticnanoparticles disposed within the polymer substrate.

Example 52 includes the subject matter of any of Examples 45-50, whereinthe functionalized beads of the first type include magneticnanoparticles disposed on a surface of the polymer substrate.

Example 53 is a method of detecting a complex including an analyte, themethod comprising: (a) contacting a sample in a solution with apopulation of functionalized beads of a first type, which are magneticfunctionalized beads and are functionalized to include a first moietythat associates with an analyte under suitable conditions, (b)contacting the sample solution with a population of functionalized beadsof a second type, which comprise a polymer substrate impregnated with afluorescent material, and are functionalized to include an unlabeledmoiety that associates with the analyte under suitable conditions,contact resulting in formation of a complex including one of the firsttype of functionalized bead, the analyte, and one of the second type offunctionalized bead, (c) illuminating the complex with incident lightthat excites fluorescence within the functionalized beads of the secondtype, and (d) detecting the complex including the analyte by analyzingfluorescence emitted by the functionalized beads of the second typeassociated with the analyte in the complex.

Example 54 includes the subject matter of Example 53, further includingapplying a magnetic field gradient to the sample solution aftercontacting the sample with the population of functionalized beads of thefirst type.

Example 55 includes the subject matter of Example 54, wherein applyingthe magnetic field gradient to the sample solution is performed aftercontacting the sample solution with the population of functionalizedbeads of the second type.

Example 56 includes the subject matter of any of Examples 53-55, furtherincluding concentrating the sample solution after contacting the samplesolution with the population of functionalized beads of the second type,before detecting the complex.

Example 57 includes the subject matter of any of Examples 53-56, furtherincluding agglomerating a plurality of functionalized beads of the firstand second types, after contacting the sample solution with thepopulation of functionalized beads of the second type, before detectingthe complex.

Example 58 includes the subject matter of any of Examples 53-57, furtherincluding dehydrating the sample solution before detecting the complex.

EQUIVALENTS

Having thus described several illustrative embodiments, it is to beappreciated that various alterations, modifications, and improvementswill readily occur to those skilled in the art. Such alterations,modifications, and improvements are intended to form a part of thisdisclosure, and are intended to be within the spirit and scope of thisdisclosure. While some examples presented herein involve specificcombinations of functions or structural elements, it should beunderstood that those functions and elements may be combined in otherways according to the present disclosure to accomplish the same ordifferent objectives. In particular, acts, elements, and featuresdiscussed in connection with one embodiment are not intended to beexcluded from similar or other roles in other embodiments. Additionally,elements and components described herein may be further divided intoadditional components or joined together to form fewer components forperforming the same functions.

The foregoing description of example embodiments has been presented forthe purposes of illustration and description. It is not intended to beexhaustive or to limit the present disclosure to the precise formsdisclosed. Many modifications and variations are possible in light ofthis disclosure. It is intended that the scope of the present disclosurebe limited not by this detailed description, but rather by the claimsappended hereto. Future filed applications claiming priority to thisapplication may claim the disclosed subject matter in a differentmanner, and may generally include any set of one or more limitations asvariously disclosed or otherwise demonstrated herein.

What is claimed is:
 1. A method of detecting a complex including ananalyte, the method comprising: (a) contacting a sample in a solutionwith a population of functionalized beads of a first type, which aremagnetic functionalized beads and are functionalized to include a firstmoiety that can specifically associate with an analyte under appropriateconditions; (b) contacting the sample solution with a population offunctionalized beads of a second type, which are functionalized toinclude a second moiety that associates with the analyte under suitableconditions, contact resulting in formation of a complex including one ofthe first type of functionalized bead, the analyte, and one of thesecond type of functionalized bead; and (c) detecting the complexincluding the analyte by detecting magnetic fields produced by themagnetic functionalized bead and by detecting the functionalized bead ofthe second type associated with the analyte in the complex.
 2. Themethod of claim 1, further including disposing the sample solutionincluding the complex over a substrate that includes at least oneoptically detected magnetic resonance (ODMR) center formed in thesubstrate; exciting electrons within the at least one ODMR center from aground state to an excited state with incident light; applying a biasmagnetic field on the complex; and generating a microwave field incidenton the at least one ODMR center, the microwave field includingfrequencies that correspond to ground state transitions in the at leastone ODMR center, wherein detecting the complex including the analytefurther includes analyzing light emitted by the at least one ODMRcenter, characteristics of the emitted light being influenced by themicrowave field and by the magnetic functionalized bead associated withthe analyte in the complex.
 3. The method of claim 2, wherein the atleast one ODMR center is a nitrogen-vacancy center in a diamond lattice.4. The method of claim 3, wherein the at least one ODMR center is formedin an upper surface of the substrate.
 5. The method of claim 4, whereinanalyzing light emitted by the plurality of ODMR centers includesimaging the emitted light.
 6. The method of claim 1, further includingapplying a magnetic field gradient to the sample solution aftercontacting the sample with the population of functionalized beads of thefirst type.
 7. The method of claim 1, wherein the population offunctionalized beads of the first type and the population offunctionalized beads of the second type are added to the sample solutionsequentially.
 8. The method of claim 1, wherein the functionalized beadsof the second type are fluorescent functionalized beads, and the methodfurther includes illuminating the complex with incident light thatexcites fluorescence within the functionalized beads of the second typeand fluorescence imaging of the complex.
 9. The method of claim 1,wherein the functionalized beads of the second type are magneticfunctionalized beads, including a magnetic property distinguishable fromthe functionalized beads of the first type.
 10. The method of claim 1,further including applying a magnetic field gradient to the samplesolution after contacting the sample solution with the functionalizedbeads of the first and second types.
 11. The method of claim 10, furtherincluding varying the magnetic field gradient applied to the samplesolution.
 12. The method of claim 1, further including concentrating thesample solution after contacting the sample solution with the populationof functionalized beads of the second type.
 13. The method of claim 1,further including agglomerating a plurality of functionalized beads ofthe first and second types, after contacting the sample solution withthe population of functionalized beads of the second type, beforedetecting the complex.
 14. The method of claim 1, further includingdehydrating the sample solution after disposing the sample solution overthe diamond substrate.